(1) First, we compared RNA expression of different PDE subtypes in rabbit SANC and ventricular myocytes (VM). Total RNA was reverse transcribed to generate complementary DNA (cDNA), and relative abundance of cDNA from 9 different PDE transcripts was measured with qPCR. PDE3A, PDE4B and PDE4D were the major PDE subtypes expressed in both rabbit SANC and VM. We verified expression of major PDE subtypes (PDE3A, PDE4B and PDE4D) at the protein level in the rabbit SA node and ventricle using western blot. Consistent with the qPCR data, PDE3A protein was more abundant in the rabbit ventricle than in SA node. There was comparable expression of PDE4B protein in the SA node and ventricle, while expression of PDE4D protein was more abundant in the SA node than in ventricle. To test our first hypothesis we used phosphorylation of phospholamban (PLB) at Ser16 site as a marker of cAMP/PKA-dependent protein phosphorylation in SANC. Specific PDE3 inhibitor, cilostamide (Cil, 0.3 mkmol/L), or a PDE4 inhibitor, rolipram (Rol, 2 mkmol/L), increased PLB phosphorylation by 20%, but the combination of Cil+Rol increased PLB phosphorylation by 110%, an effect similar to that (140%) produced by broad spectrum PDE inhibitor IBMX. L-type Ca2+ current (ICa,L) ensures LCR existence, providing Ca2+ available for pumping in the SR. Cil or Rol alone increased the amplitude of ICa,L by 60% and 4%, respectively, while (Cil+Rol) or IBMX increased ICa,L by 100%. Cilostamide increased the spontaneous SANC firing rate (perforated patch-clamp) by 20% (from 14813 to 17513 beat/min, n=8), while rolipram produced no acceleration of spontaneous firing at 2, 20 or 100 mkmol/L. Similar to IBMX combination of (Cil+Rol) increased the spontaneous SANC beating rate by 50% (from 1348 to 19711 beat/min, n=9), the effect was due to a marked increase in the LCR number, size and decrease in the LCR period that predicted the concomitant decrease in the spontaneous cycle length. When RyR were disabled by ryanodine and LCRs were abolished, both IBMX and (Cil+Rol) failed to accelerate DD rate or increase SANC firing rate indicating key role of Ca2+ cycling for PDE-dependent control of spontaneous beating. We conclude that both PDE3 and PDE4 regulate spontaneous SANC firing, and a crucial role of PDE4 is revealed only when PDE3 and PDE4 are concurrently inhibited. Thus, synergism of combined (PDE3+PDE4) inhibition suppresses basal cAMP/PKA-dependent PLB phosphorylation and reduces ICa,L amplitude to decrease RyR Ca2+ release, prolong the LCR period and limit the spontaneous SANC firing rate. (2) To test our second hypothesis we studied changes in spontaneous firing of freshly isolated rabbit SANC produced by specific PKC inhibitors GF109203X or calphostin C. When PKC activity was suppressed by GF109203X or calphostin C there was suppression of SR Ca2+ cycling and spontaneous beating of SANC stopped. Specifically, GF109203X decreased the LCR size (from 5.80.3 to 2.80.3mkm) and number per each spontaneous cycle (from 1.40.2 to 0.70.1) and increased the LCR period, i.e. the time from the prior AP-induced Ca2+ transient to the subsequent LCR. The increase in LCR period during PKC inhibition predicted an increase in the spontaneous cycle length. All effects of GF109203X were reversed upon washout. Since Ca2+ cycling in SANC is critically dependent on ICa,L, which contributes to the AP upstroke and modulates the SR Ca2+ content, we studied effects of GF109203X and calphostin C on ICa,L. PKC inhibition by both inhibitors markedly suppressed ICa,L amplitude, strongly suggesting that modulation of ICa,L could be one of the major targets of basal PKC activation in rabbit SANC. PKC could be activated by ubiquitous enzyme phospholipase C (PLC), which plays a key role in Ca2+ signaling in numerous cell types. PLC could be activated by multiple pathways, including the cAMP mediator, Epac, or by epidermal growth factor receptor (EGFR) activation. Activation of Epac signaling by cpTOME, however, was without effect on the spontaneous SANC beating rate, ruling this pathway out. Whether EGFR is present in rabbit SANC or is involved in the regulation of cardiac pacemaker function is unknown. We discovered that both EGFR and PLC (assessed by RNA-sequencing) are expressed in rabbit SANC. Specific EGFR inhibitor AG1478 decreased the DD rate and spontaneous SANC beating rate (perforated patch-clamp recordings). EGFR inhibition by AG1478, in a time-dependent manner, suppressed LCRs (confocal microscopy, Ca2+indicator Fluo-3), i.e. decreased average LCRs size, number per each spontaneous cycle and prolonged the LCR period When PLC activity was suppressed by PLC inhibitor U-73122 spontaneous firing of intact rabbit SANC stopped, indicating a key role of basal EGFR-PLC activity for cardiac pacemaking. We conclude that EGFR-activated PLC-dependent control of spontaneous SANC firing might work through basal PKC activation, which is obligatory for normal automaticity of cardiac pacemaker cells.